PROJECT SUMMARY/ABSTRACT In type 2 diabetes (T2D), insulin-stimulated blood flow to skeletal muscle is markedly blunted which significantly limits glucose uptake, thus contributing to impaired glucose homeostasis. A detailed understanding of the precipitating factors and mechanisms underlying the defects in vasodilator actions of insulin is critical for the development of therapeutic strategies aimed at improving glycemic control and protecting against cardiovascular disease. Based on our prior work and most recent preliminary data, we propose that in hyperglycemic T2D patients, protein kinase C (PKC) activation drives the upregulation of endothelin-1 (ET-1) and consequent impairment in insulin-induced dilation. Furthermore, we hypothesize that increased vascular exposure to shear stress, associated with physical activity, mitigates these toxic molecular effects of hyperglycemia on endothelial cells and lead to substantial improvements in insulin-induced dilation in T2D. Specifically, we will test the overarching hypothesis that endothelial PKC activation mediates the upregulation of ET-1 and impairment in insulin-induced dilation in patients with T2D, a defect that can be corrected with increased physical activity and shear stress. In Aims 1 and 2, ex vivo functional studies will be performed in isolated visceral resistance arteries from obese T2D and obese non-T2D patients undergoing Roux-en-Y gastric bypass surgery. Through gain- and loss-of-function experiments, we will examine the role of PKC activation in mediating impaired insulin-induced dilation in arteries from T2D patients as well as the role of hyperglycemia and shear stress in modulating insulin-induced dilation. In Aim 3, we will perform a clinical study in patients with T2D to determine the effects of increased walking and shear stress on insulin-stimulated leg blood flow. In particular, we will test the hypothesis that increased walking for 8 weeks decreases vascular PKC activation and ET-1 production, thus leading to an improvement in insulin-stimulated leg blood flow. Leg blood flow via Doppler ultrasound will be assessed during a hyperinsulinemic-euglycemic clamp. Skeletal muscle biopsies will be performed for vascular phenotypic characterization. Furthermore, we will determine if increased leg vascular shear stress using a non-exercise stimulus (i.e., leg heating intervention for 8 weeks) recapitulates the beneficial vascular effects of increased walking. Targeting PKC activation and ET-1, pharmacologically or through an increase in shear stress, may be key for correction of vascular insulin resistance and ultimately improvement of metabolic and cardiovascular outcomes in patients with T2D. Indeed, our research team is poised to move cardiovascular and diabetes research forward in an area currently receiving little attention, despite its importance and clear need for investigation.